**1. Introduction**

Leishmaniasis is a major public health problem affecting the poor population of the world, mainly in the developing countries. The disease is endemic in 97 countries with 70,0000 to one million new cases per year [1,2]. Visceral leishmaniasis (VL), caused by the protozoan *Leishmania donovani*, is the most severe type, with frequent outbreaks and a greater mortality potential. In 2018, more than 95% of new cases reported to World Health Organization (WHO) occurred in 10 countries, including India [1,3]. Due to the lack of a vaccine and effective vector control, management of VL relies exclusively on a handful of chemotherapeutic agents, but most of the therapeutics, including pentavalent antimonials, miltefosine, and liposomal amphotericin B, are associated with serious drawbacks, such as being toxic and expensive, with a declining efficacy pertaining to an increase in the occurrence of resistance [4–6]. Therefore, there is a need to explore new safe, effective, and affordable treatment options for VL.

The antimalarial drug artemisinin and its derivatives have been found to also be effective against non-malarial parasites, such as *Leishmania*. There are several in vitro and in vivo studies demonstrating the antileishmanial activity of artemisinin and its derivatives with a high safety index [7–9]. As far as the mechanism of action is concerned, artemisinin and its derivatives have been reported to cause programmed cell death in *Leishmania* promastigotes by a loss of mitochondrial membrane potential, enabling externalization of phosphatidylserine, DNA fragmentation, and cell cycle arrest at the sub-G0/G1 phase [10]. The drug also works by the restoration of normal nitric oxide (NO) production by infected macrophages, initially impaired due to infection with *Leishmania* parasites [11–13]. Further, studies in mice suggest that administration of artemisinin results in the generation of iron-artemisinin adducts, which causes clearance of intracellular amastigotes [14]. However, antileishmanial activities and the possible mechanism of resistance to artemisinin in *Leishmania* parasites have been poorly explored. An understanding of the mechanisms of drug resistance in *Leishmania* is vital to protect existing drugs and for the development of new ones [15]. Drug-resistant parasites apply various strategies in order to survive under drug pressure, such as reduced drug uptake, active drug efflux, alteration of the drug targets, inactivation of drugs, etc. [16–22]. Various transcriptomic studies of drug-sensitive vs. -resistant parasites revealed that a number of genes have altered expression in drug-resistant parasites. Our group has previously shown by microarray analysis that approximately 3.9% and 2.9% of the total *Leishmania* genome representing various functional categories, such as metabolic pathways, transporters and cellular components among others, were differentially modulated (>2 fold) in experimentally selected miltefosine- and paromomycin-resistant lines, respectively [23,24].

Whole-genome sequence (WGS) analysis is another important tool used to detect mechanisms of drug resistance in *Leishmania*. It was earlier reported that in the absence of transcriptional control, *Leishmania* parasites have evolved mechanisms to alter mRNA levels by increased gene dosage through gene amplification, gene deletion, and aneuploidy in order to adapt to stress conditions, such as drug pressure [25–28]. The genome sequence of *Leishmania* field isolates from the Indian sub-continent revealed gene copy number variation (CNV) to be associated with susceptibility to sodium stibogluconate (SSG) [29]. Similarly, aneuploidy has been observed in the context of antimony, methotrexate, and nelfinavir resistance; however, the link between aneuploidy and drug resistance was circumstantial [25,26,30–32]. Additionally, single-nucleotide polymorphisms (SNPs) in drug targets or key enzymes constitute another strategy to survive under drug pressure. The acquisition of an inactivation mutation in the *L. donovani* miltefosine transporter gene (*Ld*MT) and/or its β-subunit (LdRos3) was reported to increase miltefosine resistance in both in vitro and in vivo studies as well as in clinical isolates [33–38].

Artemisinin resistance in malaria is associated with SNPs on chromosome 10, 13, and 14, and non-synonymous SNPs in the propeller domain of a kelch gene located on chromosome 13 [39,40]. Analysis of the transcriptome of *Plasmodium falciparum* isolates revealed a higher expression of unfolded protein response (UPR) in artemisinin resistance. Previously, we explored the mechanism of artesunate (a derivative of artemisinin) resistance in *Leishmania* parasites and showed that artesunate resistance in *Leishmania* is associated with parasite virulence, host immune modulation, and unfolded protein responses [41]. In the present study, the genome and transcriptome of artesunate-sensitive vs. -resistant *Leishmania* parasites were analyzed using next-generation sequencing (NGS) and single-color DNA microarray technology, respectively. Analysis of the genome structure and modulated gene

expression identified several genes/pathways, which were further validated for their role in the selection of artesunate resistance in *Leishmania*. Expression analysis of Heat shock protein 70 (Hsp70) and Aquaglyceroporin 1 (AQP1) was validated in K133WT and K133AS-R cell lysate. In view of the important roles of ATP-binding cassette protein (ABC) transporters and the AQP1 gene in drug resistance in *Leishmania*, their roles were explored in artesunate resistance using respective inhibitors. Based on the analyses, a model was predicted for artesunate resistance in *Leishmania*.
